1. Field of the Invention
This invention relates generally to microfabrication processes for microfluidics. More specifically, the invention is a system and method of microfabrication that use planar, thin-film microfabrication techniques from which microfluidic and microelectronic components are combined on a substrate to perform bioanalytical microfluidic operations.
2. Description of Related Art
An increasing need for the detection and quantification of biological molecules in medicine, biochemistry, and biology has driven a rapid expansion in the analysis of biomolecules. Innovations in bioanalytical chemistry have enhanced the ability to characterize a wide range of analytes, including metabolites, neurotransmitters, nucleic acids, carbohydrates, peptides and proteins. The continued development of new tools and techniques that improve sensitivity, selectivity, speed and throughput in biological analysis, while reducing cost per assay, are critical in clinical diagnosis, medical research, and other disciplines in the life sciences.
A major interest in bioanalytical chemistry is the separation and identification of proteins. The distribution of proteins in biological materials is sensitive to cellular conditions, and consists of proteins having abundances that are dependent on age, disease state(s), and environmental conditions (e.g., nutrients, medicines, temperature, stress, etc.). Marker proteins, whose expressions change during the progression of a disease, have been associated with certain human ailments such as cancer, Alzheimer's disease, schizophrenia, and Parkinson's disease, to name a few. Measurements of such target proteins are becoming increasingly important in clinical assays for human disorders and disease.
Quantitative analysis of protein expression profiles has been proposed as a means to diagnose the overall state (e.g., wellness) of the biological system from which they were obtained. In order to take advantage of this extremely promising diagnostic potential, appropriate methodologies must be developed to rapidly separate, identify, and quantify target proteins in various samples of interest, including body fluids, tissues, and cells.
Unfortunately, protein analysis is an extremely challenging task because of the sheer number of proteins in biological systems and their dynamic nature. There are vastly different concentrations of the various proteins; for a given cell, the abundance of different proteins may vary by over a factor of a million, while in the blood the dynamic range of protein concentrations may be greater than ten orders of magnitude. Moreover, numerous interactions occur among proteins and other ligands, and expressed proteins are often further modified by reactions such as phosphorylation, glycosylation, carbamylation, deamidation, and truncation. Considering that current studies place the number of genes in the human genome to be about 22,000 and that there are many more proteins than genes, the enormity of the analytical problem is clearly evident. It has been estimated that there are approximately 1,500,000 different proteins expressed in humans. Clearly, sophisticated new analytical techniques with extremely high peak capacities and very large dynamic detection ranges are needed.
Currently, the most popular method for separating a large number of proteins is two-dimensional (2-D) gel electrophoresis. Using this technique, proteins are separated in one dimension by isoelectric point (pI) and in a second dimension by size. Although 2-D gel electrophoresis can resolve more than 1,000 proteins in an analysis, it has some serious limitations. First, the resolving power of 2-D gel electrophoresis is insufficient to separate the numerous proteins that may be important in the profile; i.e., 1,000 proteins compared to a possible 1,500,000. Second, the reproducibility of the technique is insufficient, making it difficult to detect differences in protein expression reflected in two different gels. Third, this technique is time-consuming (i.e., as much as several days) and labor-intensive. Fourth, various stains must be used to visualize the spots for digitization by a scanner or for excision for subsequent mass spectrometry (MS). These stains have variable sensitivities to protein structure and mass present in the spot and, therefore, are not reliably quantitative.
Because of the difficulties and limitations encountered when using 2-D gel electrophoresis for protein analysis, researchers are striving to develop alternative approaches. Recent noteworthy developments have been reported by several groups. An alternative to 2-D gel electrophoresis is coupling liquid-phase isoelectric focusing (IEF) to nonporous reversed phase high performance liquid chromatography (LC), which can be detected using MS. The analysis of breast epithelial cells in two selected ranges of pI values led to the detection of ˜110 proteins. Important differences in protein levels were observed between malignant and normal cell lines.
Others have developed 2-D LC systems coupled with MS for the analysis of complex protein mixtures. 2-D LC was performed in a so-called “biphasic” column containing reversed-phase packing followed by a strong cation exchanger. A 3-phase column, having an additional segment packed with reversed phase particles, enabled sample desalting on column. The 3-phase LC system provided a greater number of protein identifications than the “biphasic” column in analyzing a protein mixture from bovine brain.
Still others have reported 2-D liquid-phase separations using capillary IEF combined with capillary reversed-phase LC. A micro injector system provided the interface between the two separation dimensions. This system was evaluated on a Drosophila salivary gland soluble protein fraction and gave peak capacities as high as ˜1,800. An important advantage of this system is that the IEF step provides 50-100 fold sample concentration, which may help in the characterization of less-abundant proteins. While the analysis times for this system were an improvement over the several days often required for 2-D gel electrophoresis, the ˜8 h separation time is still slower than desired.
There is considerable interest in miniaturization of 2-D separation systems for protein and protein digest analysis. One group developed a microfluidic system that combined IEF with a series of denaturing capillary gel electrophoresis (CGE) channels. While promising initial results were obtained on a 3-protein mixture, this system required manual removal of buffer reservoirs and peeling off a polymer layer between the IEF and CGE step. Others developed a polymer microfluidic system for integrating IEF with CGE. However, the IEF step was carried out in a separate apparatus, and the small IEF gel strip had to be transferred manually to the microfluidic CGE separation platform. The separation of a mixture of 6 proteins was shown. Another group created a plastic microdevice with an IEF channel interfaced with 10 sieving matrix filled CGE channels. A 2-D analysis of 5 model proteins was done, and a maximum peak capacity of 1,700 was projected from the results. Nevertheless, the wide spacing (1 mm) and small number (10) of electrophoresis channels in this format limits performance and complicates detection.
One group recently developed a microchip system for 2-D separation of protein digests. In this micromachined platform, a micellar electrokinetic capillary chromatography separation provided the first dimension, while capillary zone electrophoresis (CZE) was the second separation dimension. Analyses of serum albumin, ovalbumin and hemoglobin tryptic digests were performed. While only 50-80 baseline-resolved fragments were observed in these separations, a maximum peak capacity of ˜4,000 peptide fragments was projected for this approach, based on the widths of the peaks in each of the separation dimensions. Importantly, the analysis time for this approach was very fast (10-15 min), illustrating one of the benefits of miniaturized separation methods.
These recent advances in multidimensional liquid-phase separations have addressed some of the shortcomings of conventional protein analysis technologies, such as speed, automatability, and convenient interfacing with MS detection. However, a critical need still exists for new technologies for the analysis of complex protein mixtures, especially platforms that integrate sample pretreatment and detection schemes with multidimensional separations.
Miniaturization in chemical separations with many of the same technologies used in integrated circuit (IC) or “chip” manufacture got its start over 25 years ago and has grown substantially since renewed interest was sparked by the development of planar microfabricated CE substrates in 1992. While miniaturization has the potential to revolutionize chemical separation methods as it did with integrated circuits, thus far the impact of microfabrication on separations has been modest.
While the initial microchip electrophoresis experiments were carried out exclusively on glass substrates, more recent efforts have also focused on the use of easily replicated and low-cost plastic or elastomeric materials. Poly(methylmethacrylate) (PMMA) microchannel systems, which have desirable optical and mechanical properties, were among the first polymeric substrates evaluated for microfluidic analyses. The ease of fabrication of microchannel systems in poly(dimethylsiloxane) (PDMS) made this material another appealing candidate substrate for microchip analytical platforms. A number of other polymeric microchip substrates have also been studied more recently. While typically providing simplified and low-cost device fabrication, polymeric substrates are disadvantageous in terms of compatibility with conventional Si processing methods that may require elevated temperatures, for example in thin-film deposition, which would hamper the direct integration of planar polymeric devices with some electronics, detection instrumentation, etc. Moreover, polymeric materials tend to be more compatible with stacked, rather than planar thin-film designs. To date, little success has been seen in fabricating functional microfluidic systems on silicon substrates.
The miniaturization of pumping methods for microfluidic systems has been pursued in several different ways, including the use of surface properties, pressure-actuated flexible membranes, or electroosmosis. The programmed control of surface temperature in thermocapillary pumping, or surface free energy using tailored self-assembled monolayers can enable liquid flow, but these approaches suffer from a significant level of device fabrication and surface modification complexity. Electrolysis-driven displacement of fluids and micromachined membranes has also been used for pumping. However, in the former approach, the electrolysis solution directly contacts the liquid being pumped, potentially causing contamination, while the latter method is complicated by the need to microfabricate a thin membrane for each pump.
Valves and micropumps that are driven by the actuation of flexible elastomeric membranes in controlled sequences have also been demonstrated. For these modules, the need for external pressure and vacuum sources to actuate the membranes makes the entire system difficult to miniaturize. Moreover, the use of PDMS membranes in direct contact with pumped fluids is problematic for LC, because PDMS acts as a hydrophobic stationary phase in chromatography.
Recent efforts to use electroosmosis to pump liquids in microchannels have also been reported. Issues not yet addressed with this form of pumping include challenges associated with reproducible etching of very shallow channel arrays and reliable thermal attachment of a cover plate to create large bundles of integrated microcapillaries to achieve suitable pressures. Hence, it would be an advantage over the state of the art to have pressure-driven micropumps that are easily integrated with both planar electronics and microfluidics.
One of the challenges with increasingly parallel microfluidic arrays is the two-dimensional nature of planar microchip systems. Some efforts have been directed at creating three-dimensional, layered microfluidic manifolds, but the initial attempts were quite cumbersome in terms of both fabrication and fluidics. More sophisticated recent work has utilized multiple-layer devices to create fluidic manifolds with increased liquid routing complexity. However, these systems utilize via holes through layers that can contribute to dead volume, and use materials such as PDMS that have poor stability in many solvents and reduced compatibility with planar Si micromachining processes. Thus, new approaches for creating complex sample handling manifolds for parallel analysis are still needed.
The systems described above demonstrate that there has been important progress in integration. Nevertheless, further improvements in terms of scaling down to microchannel dimensions in devices, and miniaturization and integration of detection are still needed. Moreover, these integrated microsystems could greatly enhance biochemical analysis. In summary, the full utility of miniaturization in separation-based chemical analysis will only be achieved when all components are integrated and reduced to a small size scale. Hence, what is needed is the development of fabrication techniques that will enable the creation of microfluidic systems which are compatible with conventional planar integrated circuit manufacturing processes.
The present invention is a system and method for performing rapid, automated and high peak capacity separations of complex protein mixtures through the combination of fluidic and electrical elements on an integrated circuit, utilizing planar thin-film micromachining for both fluidic and electrical components.
These and other objects, features, advantages and alternative aspects of the present invention will become apparent to those skilled in the art from a consideration of the following detailed description taken in combination with the accompanying drawings.
Reference will now be made to the drawings in which the various elements of the present invention will be given numerical designations and in which the invention will be discussed so as to enable one skilled in the art to make and use the invention. It is to be understood that the following description is only exemplary of the principles of the present invention, and should not be viewed as narrowing the claims which follow.
The present invention is a system and method for integrating microfluidics directly onto electronic systems, thereby making it possible that logic and electric power elements can be built into a microfluidic analysis system.
One important advantage of using planar, silica-based microfabrication processes is the existing fabrication tools. This point is fundamental to the present invention. The microelectronics industry has spent tens of billions of dollars developing integrated circuit (IC) fabrication technology, making it some of the most advanced and precise equipment in the world. These available tools make research and development much easier, and allow compatible processes to be done cheaply and on a large scale at silicon foundries around the globe.
Another advantage of using IC fabrication technology for the present invention relates to the methodology that has been developed. Much of the burden of complex electronic circuit design in the microelectronics industry has been shifted to specially designed computer aided drafting (CAD) programs. These programs contain what will be termed a “toolbox” to help the designer. In the toolbox are designs for hundreds of active devices and functional groups. For instance, if a common logic element or voltage converter is needed in a design, a user can simply select from the toolbox and drop it into the design. CAD programs also include automatic wire routing routines, design rules for placement and size requirements, and performance models that provide very accurate predictions of how a circuit will work before it is ever made in silicon. CAD tools allow teams of designers to work on the same large circuit divided into functional groups. Successful design using CAD tools relies on very robust fabrication processes and devices. A transistor must look and act the same anywhere on a chip.
In the present invention, the design of complex microfluidic circuitry can follow much of the same approaches used to do CAD layout in microelectronics. In fact many of the existing CAD programs can be adapted to allow for user-specified devices and design rules. The first step in building a microfluidics toolbox is identifying robust designs for active microfluidic components including fluid pumps, separation devices, and detectors. The second step is identifying design rules for spacing and widths of fluid channels for routing and connecting, including branching elements. Just like in microelectronics, it is very important that active devices and connections have the same performance no matter where they are placed in an on-chip network. When reproducible designs can be fabricated and well characterized, performance models can then be created to analyze a microfluidic circuit design before it is ever fabricated.
A variety of designs and mechanisms have been created for the active components of a microfluidic system. Also considered was how robust and reproducible these active devices could be made so as to fit into a CAD toolbox.
For example, pumping based on multiple layers of elastomeric membranes is not amenable to the flat, planar micromachined construct of the present invention. Of those pumping systems that are compatible with planar Microsystems technology, some require considerable levels of fabrication complexity (membrane-based actuation) or significant post-fabrication surface modification (surface property-based methods). Accounting for these considerations, electroosmotic pumping is the best-suited approach for this toolbox of the present invention because of its fabrication simplicity and ease of integration in a planar, flat format.
For networks and separators, the use of stacked, layered systems with through-holes is poorly suited to the flat, planar constraint of the toolbox. Moreover, while conventional, single-layer approaches provide some level of complexity, they are limited by the inability to provide complex fluidic routing where channels can cross over one another without interference. Thus, the microfluidic network created for this toolbox must improve over existing techniques to allow complex, crossing arrays of channels within a planar system.
Regarding detectors, it is noted that the most sensitive method for detecting the presence of a biological species of interest is to attach a fluorescent label and measure it optically using a microscope. For a highly complex on-chip fluidic test platform with many test points, using this kind of optical detection system is unreasonable. In order to have very high sensitivity optical detection for an on-chip system, it would be ideal to efficiently route light signals across a chip from a point of detection to an on- or off-chip detector away from a sample. This requires the use of optical waveguides.
Another method of detection that fits in with the planar design philosophy of the present invention is the direct electrical measurement of biological species in a microfluidic channel. This can be done by attaching electrodes across a channel and monitoring the impedance of a fluid as it flows by the electrodes. Biological material passing through the detection window will be indicated by a change in impedance. These measurements can be very sensitive and are aided by the small geometry of microfluidic channels.
Having shown what the present invention is intended to accomplish through the use of a toolbox, a typical fabrication process of the present invention is now described in detail. One of the keys to achieving a truly integrated on-chip sensor system was the development of a novel fabrication method of the present invention that can be used on a variety of surfaces (i.e., semiconductors, insulators, polymers, ceramics, metals and glasses). This fabrication method must enable the creation of hollow channels for fluid manipulation, waveguides for routing optical detection information, and metallic lines and pads for routing electrical signals. In the interest of economics, it is also an important aspect of the present invention that any new microfabrication techniques not stray too far from standard procedures used in the microelectronics industry.
The first step of the present invention is to select a suitable substrate material. So as not to stray from existing techniques for creating integrated circuits, a silicon substrate will be used in the following example. Nevertheless, it should be understood that any suitable substrate material known to those skilled in the art of integrated circuit fabrication can also be used in the present invention. As explained, these substrate materials include semiconductors, insulators, polymers, ceramics, metals and glasses.
The next step is to create hollow tubes by surrounding a sacrificial core with silicon dioxide or silicon nitride. The sacrificial core is then removed with etching.
This fabrication process of the present invention is depicted in
In one embodiment of the present invention, a substrate 30 is coated with silicon dioxide and/or nitride layers 32 using plasma enhanced chemical vapor deposition (PECVD). This process takes place at approximately 250° C. In development of the present invention, silicon, quartz, and glass substrates have been used. Tolerance to the temperatures used in the vapor deposition process limits the materials that can be used for the substrate. In another embodiment, it is suggested that other PECVD compatible solid films such as amorphous silicon can also be used. Likewise, evaporated dielectric thin films like alumina and silicon monoxide can be used in other embodiments.
Next, a thin layer of sacrificial material 34 is then deposited and defined into a thin line using photolithography and etching techniques.
It is observed that a variety of sacrificial materials may be used, including photosensitive polymers and metals. What is important is that the sacrificial material be capable of being removed through some process, such as acid etching, without damaging the underlying substrate or other layers of materials on the substrate, if any.
An overcoat layer of PECVD oxide 36 or nitride is then grown which covers the sacrificial material 34. The conformal nature of this process is important to ensure that the sacrificial material 34 is completely enclosed.
The final step of the process is to expose the sacrificial material 34 to an etch from either end of the channel 38. Upon completion of the acid etch, the result is a hollow tube 40 with walls composed of either silicon dioxide or silicon nitride.
The process above describes the creation of a single, straight, hollow tube. However, one of the important advantages of the present invention is the ability to create tubes that can bend, tubes that can cross over other tubes without being in communication, tubes that can intersect and join with other tubes, and the creation of multiple parallel tubes, to name just a few. Thus, like a complex transistor circuit having multiple electrical connections and layers all meeting in the same location in a very precise manner, the present invention is able to create complex interactions and intersections of tubes for the flow of fluids.
A number of sacrificial materials have been investigated in the context of the fabrication process described above. These sacrificial materials include aluminum, SU8 (a photosensitive epoxy), and photoresist. Aluminum is most quickly removed using a nitric and hydrochloric acid etching solution while SU8 and photoresist are removed using a sulfuric acid and hydrogen peroxide solution. The different sacrificial materials result in different shaped hollow core cross sections as illustrated in
The hollow channels 40, 42, 44 shown in
The process can also be used to produce a much wider channel 46 as shown in
In order for structures made using the new planar, thin-film technology of the present invention to be useful for fluid and light guiding, the channels must have smooth inner walls, be of reasonable length, and mechanically strong. The first criterion is important to prevent optical scatter or interruptions in fluid flow and is met by the conformal CVD coating as evident in SEM micrographs of the channel structures. Experiments to determine ultimate channel length and strength were conducted and were compared to physical models.
Because hollow structures are formed through the chemical etching of a sacrificial layer, the ultimate channel length and fabrication time will be dependent on this chemical process. Etch times were investigated for aluminum sacrificial cores 700 nm thick patterned on silicon. Core width varied between 10 and 300 μm. A single layer of silicon dioxide 3.0 μm thick was deposited over the aluminum, the silicon substrate was cleaved, and then the samples were placed in an aqua regia (3:1 mixture of hydrochloric and nitric acid) solution. Samples were periodically removed from the acid solution, and the amount of aluminum that was etched was measured using an optical microscope.
l(t)≈√{square root over (2knDcot)} EQUATION 1
where l(t) is the length of the channel etched in a given time, kn is a constant relating to the geometry of the channel, D is the diffusion coefficient for etchant through the channel, and co is a constant relating the concentration of the most critical component of the etch solution. Curves with a square root dependence of etched distance versus time were fit to each of the data sets in
In order to determine the ultimate mechanical strength of the hollow structures, a finite-element analysis was done using a commercially available software package (ANSYS 6.0). To provide the necessary stress and strain constants to the software, a set of experiments was also done. The results of the model indicate that the critical failure pressure for a hollow channel can be given by the simple expression:
where St is the tensile strength of the overcoat material, th is the thickness of the overcoat layer, and w is the width of the channel. This simple equation reveals the functional dependence of the pressure on the width and thickness, and agrees within 10% of the values calculated using the finite-element simulation when th/w<1/10. Tests were done on real structures by varying their core width and overcoat thickness to confirm this expression. Results are shown in
Another aspect of the present invention with regards to the creation of integrated microfluidic devices is the ability to generate networks of fluid channels that can route liquids over the surface of a chip much like dense electrical signals are routed in integrated circuits. This requires the creation of cross-over elements and T-branches just like in macro-plumbing.
The first of these structures is shown in
Branching structures were also created using the present invention by applying a sacrificial core and photodefining it into a desired branching geometry.
To demonstrate their utility and robustness, the intersecting hollow channels shown in
Interfacing microfluidic devices to external fluid sources and reservoirs is another important consideration for an integrated system because every analytical system must interact with the macro world. A number of schemes have already been investigated that would provide large fluid reservoirs on chip and be compatible with hollow core devices.
The most successful procedure to date involves laser cutting cylinders 70 in PMMA and directly attaching the cylinders to the substrate by heating to 200° C. The bottom of the cylinder 70 melts and attaches conformally to the substrate, sealing around the hollow channels and forming a small reservoir that holds 10 μL. Pipettes or syringe needles can then be used to fill or extract liquids.
A major aspect of microfluidics is the manipulation of fluid flows in small on-chip channels. One of the most attractive ways of doing this is by using electrical forces (electroosmotic flow). An electroosmotic pumping device can be built by directing the fluid flow generated from a large number of small diameter channels from one reservoir into another. The design of such a pump is illustrated in
Implementing an electroosmotic pump with enclosed channels is implemented as follows. A sacrificial core was applied and then photodefined into the pump geometry. Conformal PECVD oxide was grown over the core, and then the sacrificial layers were removed. Pumps were made on silicon, glass, and quartz substrates using aluminum as the sacrificial material.
A pump fabricated on an SiO2 substrate with 100 channels (1 μm in width and depth each) feeding into a single 40 μm wide channel was evaluated. The pump was initially filled with a pH 9.5 carbonate buffer solution. A reservoir surrounding the small channels was filled with carbonate buffer containing 9.1 ppm rhodamine B. Voltage was applied to the pump reservoir, and the pooled buffer at the opening of the large channel was grounded, driving the electroosmotic flow toward ground. The movement of rhodamine B through the large channel was followed using a CCD to image the laser induced fluorescence signal from the compound. The 514 nm line from an Ar ion laser directed into an inverted microscope was used to excite the fluorescence. CCD images were taken at a rate of 50 Hz, and included 15.2 mm of the large channel. Flow rates were determined by the time span between the initial appearance of rhodamine B and complete filling of the imaged channel.
Waveguides were mentioned previously as being an important element of the microfluidic components. In the present invention, optical detection on microfluidic platforms will be played by ARROW waveguides. These structures enable light to be routed through liquid channels on the surface of a chip from optical sources to points of detection, and from points of detection to on-chip and off-chip optical detectors. ARROW construction requires the deposition of several alternating layers of silicon dioxide and silicon nitride of thicknesses specific to the wavelength of light to be guided. These layers surround the sacrificial core material in all dimensions.
A cross sectional view of an ARROW is shown in
Because fluorescence detection is one of the most sensitive measurement techniques for biological samples, ARROW waveguides were also filled with fluorophore containing liquids as illustrated in
This same setup was used to measure detection limits for fluorescence signals. The results are shown in
It is also desirable to integrate liquid waveguides with solid-core waveguides for routing optical pump or measurement signals. One application would be to illuminate only a very small volume of liquid inside a waveguide (femtoliters, fL) for detecting single molecules by intersecting a solid core waveguide with a liquid one as illustrated in
Taking advantage of the existing oxide and nitride layers used in constructing ARROW waveguides, the integrated structure is created as shown in
The use of impedance/conductivity measurements on the microscale has also begun to emerge in high resolution scanning systems as well as in fluid channels. Especially relevant to measuring biological agents of less than 1 μm in length has been recent work the inventors have done involving a high resolution scanning system and specially designed microprobes. To this point, probes with dimensions of approximately 10 μm have been able to produce images with resolution of approximately 10 μm. The goal is to produce probes with dimensions of less than 1 μm using micromachining to generate high-resolution images of single cells. The same techniques used to produce impedance contrast information in this scanning system can be applied to microfluidic channels.
Packaging options for microfluidic components based on planar, thin film technology are almost limitless, and each depends on the application of interest. In fact, most fluidic manipulations can be addressed by this technology. The previous sections have addressed the development of a variety of components that could comprise a microfluidic chip. This next discussion describes the design of a microfluidic device that integrates the components necessary to address a very complex application.
The area of proteomics is extremely challenging, requiring complex multidimensional approaches to separate and identify the vast number of proteins in biological samples, especially those that are present at trace levels. The planar microfluidic technology taught by the present invention allows the integration of many protein manipulation steps in a microdevice for separation and identification of complex protein samples at resolution and speed never before achieved.
As an example of the power and versatility of planar, thin film microfluidics for biomedical applications, the present invention makes possible the fabrication of a microfluidic chip that integrates the steps of extraction, concentration, separation, and identification of complex protein samples.
Two possible configurations of the overall schematic of the microfluidic layout are shown in
This two-step sample clean-up and concentration process is effected by applying voltage between sample reservoir 1 and a waste reservoir 5. Termination of sample loading and further clean-up of the bound protein sample can be accomplished by switching the voltage from sample reservoir 1 to a rinse reservoir 6 so that current flows from rinse reservoir 6 (activating electroosmotic pump 7) through monolith 4, rinsing off non-bound species to waste reservoir 5.
The protein sample is desorbed from monolith 4 by switching the voltage from rinse reservoir 6 to a desorber reservoir 8 and from waste reservoir 5 to another waste reservoir 10, allowing electroosmotic pump 9 to move desorber buffer through monolith 4, displacing the bound proteins from the monolith. The desorber solution will flow into waste reservoir 5 by pressure flow because channel 11 between reservoir 5 and waste reservoir 10 is filled with isoelectric focusing gel. As the desorbed proteins enter the T-junction of waste reservoir 5, they are drawn into the isoelectric focusing channel 11 by electrophoresis.
The isoelectric focusing channel 11 contains gel bonded immobilines that create a pH gradient along the channel to focus and concentrate proteins according to their pI values. After the proteins are focused, they are driven into numerous orthogonal gel electrophoresis channels 12 by switching the voltage from desorber reservoir 8 to buffer reservoir 15 and from waste reservoir 10 to buffer reservoir 20. Proteins will be separated according to size and charge by gel electrophoresis in channels 12. In order to maintain constant pH at the top of the CGE channels, buffer 15 will be continuously pumped by electroosmotic pump 16 through intersection point 13 into waste reservoir 17. At the end of each channel 12, the proteins will be introduced into a monolith 14 containing a bonded protein digestion enzyme. The peptides that are formed in the monolith will move from the monolith immediately into a peptide concentrating area 18 (separated merely by a conductive membrane from flowing buffer in contact with the voltage source at reservoir 20) before being released for CZE separation in channels 19. Buffer 20 will be continuously pumped by electroosmotic pump 21 through intersection points 14 into common waste reservoir 22 in order to maintain constant pH in the intersection points 14. Periodically, during concentration of peptides in the peptide concentrating area, voltage will be momentarily switched from reservoir 15 to reservoir 23 to release the concentrated peptides and initiate fast CZE separation in channels 19. During the CZE separation in channels 19, migration in the CGE channels will be stopped. By switching the voltage back and forth between reservoirs 15 and 23, proteins that migrate into the digestion monolith will be fragmented, trapped, and subsequently separated by CZE to produce peptide profiles that are characteristic of each of the proteins in the sample. These peptide digest profiles will be used in a similar way that mass spectra are used to identify compounds. Different bonded digestion enzymes can be used in different microfluidic systems to provide complementary fragmentation profiles for more definitive identification of the proteins.
Two different detection systems can be used: electrical impedance measurement of native peptides (shown schematically in
Having described at least one embodiment of the present invention, some observations are useful. For example, at least two layers of fluid routing channels will need to be constructed for most applications. This is illustrated in
It is another aspect of the present invention that it can also be used to integrate sample pretreatment and concentration processes. A microfluidic subsystem for sample clean-up and concentration will be developed as shown in
Another subsystem that can be created using the teachings of the present invention is an integrated 2-D separation, consisting of IEF followed by CGE. Development of this package will be critical to achieving high peak capacity separations. A device layout for this subsystem is illustrated schematically in
A zoom view of the intersection of the IEF channel with a few CGE channels is shown in
In the first method, the array of CGE channels will be filled through the common waste reservoir at the end of the CGE columns with pre-polymer solution (e.g. buffer solution having 4% acrylamide with a photoinitiator). Then an optical mask will be placed on top of the CGE channel array, which will allow UV radiation to polymerize the gel only in regions in the CGE channels (
An alternate, potentially simpler approach, involves filling the entire device with pre-polymer solution, adding acidic and basic immobilines to the sample and buffer reservoirs, respectively, and then migrating the immobilines into the IEF channel in an applied field. Masked UV polymerization will form gel in both the CGE and IEF channels, and then unpolymerized materials in the unexposed buffer channels will be flushed out. During immobiline migration for either approach it will be critical to minimize heat generation to prevent premature polymerization; therefore, a combination of low current and active device cooling will be utilized to avoid this issue.
The next step is to integrate CGE, protein digestion, peptide separation and fluorescent labeling. To obtain a “peptide fingerprint” of each of the separated proteins, we will integrate CGE columns with monolithic beds for protein digestion, followed by an additional separation dimension having on-column fluorescent labeling. Appropriate design of this subsystem will enable separation-based identification of each protein analyzed, providing information similar to MS detection. A layout of a device designed for optimization of this operation is depicted in
Critical to the success of a complex planar microfluidic device is a detection system that fits into the fabrication mold outlined in previous sections. The first possibility is using an optical based technique in which analytes are tagged with a fluorophore and their presence is detected by stimulating fluorescence. The application of proteomics outlined requires hundreds to thousands of optical probe points and subsequent routing of optical signals across a chip's surface.
All of the optimized components described above can be integrated into a single microfluidic system for protein analysis. However, a simpler system can be constructed and described to illustrate the basic themes of the present invention. This concept is illustrated in
It is to be understood that the above-described arrangements are only illustrative of the application of the principles of the present invention. Numerous modifications and alternative arrangements may be devised by those skilled in the art without departing from the spirit and scope of the present invention. The appended claims are intended to cover such modifications and arrangements.
This document claims priority to, and incorporates by reference all of the subject matter included in the provisional patent application docket number 05-01, having Ser. No. 60/646,184 and filed on Jan. 20, 2005. This document also claims priority to the co-pending patent application Ser. No. 10/868,475 filed on Jun. 15, 2004.
Number | Date | Country | |
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60646184 | Jan 2005 | US |
Number | Date | Country | |
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Parent | 10868475 | Jun 2004 | US |
Child | 11336646 | Jan 2006 | US |